Discovery And Evaluation Of New Drug For The Treatment Of Parkinson's Disease

Parkinson's disease (PD) is a chronic neurodegenerative disease with symptoms that include bradykinesia, akinesia, rigidity and tremor. The characteristic pathogenesis of PD is degeneration of dopaminergic neurons in substantia nigra and decrease of dopamine content in striatum. The incidence of PD is about 1% in aged person over 60. The causes of PD are still unknown. Genetic factors, infections, immunological abnormalities, aging, toxins (endogenous and exogenous) may all involve in the development of PD. In addition, oxygen free radical toxicity may play important roles in PD.Drugs currently used for PD therapy can only improve clinical symptoms but they can not retard the course of the disease. Moreover, severe side effects will appear in the long-term treatment. New drugs with good curative effect and fewer side effects are eagerly needed. Very recently, a new concept for pharmacotherapy of PD called neuroprotective strategy had been proposed.PartⅠResearch on screening of anti-Parkinson's drugsAccording to the pathogenesis of Parkinson's disease, we established 6-OHDA-lesioned SH-SY5Y cell model and promoting PC12 cell differentiation model in vitro which can display two different mechanisms in Parkinson's disease. SH-SY5Y and PC12 cells were used because both are dopaminergic cell lines. We have complete screening of 560 compounds in these two models and discovered that three new compounds have strong activity on these two models. We choose DL0705 which has the strongest activity in two different models. Further in vitro experimental results showed that DL0705 can significantly ameliorate 6-OHDA induced SH-SY5Y cell apoptosis and promote neurite outgrowth of PC12 cells, which suggest that it may be a useful agent for the prevention or treatment of PD.DL0705 (baicalein, 5, 6, 7-trihydroxyflavone) is one of the major flavonoids originally isolated from the roots of the traditional Chinese herbal medicine Huangqin, Scutellaria baicalensis Georgi. It has been widely employed for many centuries in the traditional Chinese herbal medicine as a popular antibacterial, antiviral, and anti-inflammatory agent. Several biological effects of baicalein, such as antioxidant and free radical scavenging effect, have been reported. In the following three parts, we discussed the effect of DL0705 on three different Parkinson models.PartⅡEffects of DL0705 against MPTP neurotoxicity in C57BL/6 miceHere we investigated the effect of baicalein on MPTP-induced neurotoxicity in mice. Pretreatment with DL0705(280 mg/kg, i.g.) for a week was followed by challenge with MPTP(30 mg/kg, i.p.) for five consecutive days and the subsequent behavioral, biochemical and immunohistochemical manifestations in mice were determined and compared to those in untreated mice and mice challenged only with MPTP. The present study showed that DL0705 (280 mg/kg) can significantly improve the abnormal behavior including the decrease in spontaneous motor activity and the prolongation in the pole test latent period induced by MPTP (P<0.05). The protective effect may be, in part, caused by increasing the levels of DA (P<0.01), HVA (P<0.01) and 5-HT (P<0.05) in the striatum, inhibiting the oxidative stress which displayed in the increase the GSH-Px activity (p<0.05), increasing the counts of TH immuno-reactive neurons (P<0.05) and inhibiting astroglia response (P<0.01). The results suggest that baicalein possesses potent neuroprotective activity and may be a potential anti-Parkinson's disease drug worthy of further study.PartⅢEffects of DL0705 against 6-OHDA neurotoxicity in SD rats6-Hydroxydopamine (6-OHDA) is a specific neurotoxin that selectively destroys catecholaminergic neurons. The rats received unilateral lesions of the left medial forebrain bundle (MFB) made by stereotaxic injection of 6-OHDA. Fourteen days after lesion, animals showed contralateral rotation induced by apomorphine injection. The successful rate is 60% (rotating turns≥240 r/40 min). We observed no reduction in apomorphine-induced rotations after baicalein treatment (P>0.05), but baicalein treatment can significantly improve the EMG activity (P<0.01) and there is a tendency to increase the spontaneous motor activity. The protective effect may be caused by partly increasing the levels of DA and DOPAC in striatum, increasing the counts of TH-ir neurons and inhibiting astroglia response (P<0.01), although significance was not all reached because of so serious damage. In addition, DL0705 can significantly inhibit the oxidative stress level, such as attenuate the level of MDA (P<0.05), increase the antioxidant enzyme GSH-Px (P<0.01) and SOD activity, decrease the MPO activity in the lesioned brain. All these results suggest that the effect of DL0705 was due to its strong antioxidant activity, but we also discovered that DL0705 can inhibit the peripheral velocity of nerve conduction to some extent.PartⅣEffects of DL0705 on abnormal behavior in trembling mice model induced by oxotremorineOxotremorine was injected (i.p.) to induce trembling in mice. Oxotremorine can result in a shorter latent period and a longer duration of tremor, an increased tremor intensity and gland secretory. But we didn't observed the effect of DL0705 on tremor and gland secretory (P>0.05). Oxotremorine can induce trembling through M receptor, so we can guess that cholinergic system may not be the main target of DL0705.PartⅤMechanism exploration of flavonoidsIn general, the antioxidant action of DL0705 may be its main mechanism of anti-Parkinson's disease. In this part, we discussed whether the anti-Parkinson's effect was specific to DL0705 or not. Based on the structure-function relationship and previous high throughput screening results, we chose two related compounds DL0407 and DL0609 and explored their single or combinational effect on three different Parkinson models. The results showed that these two compounds, whatever for single or combination, both displayed anti-Parkinson's effect to some extent. Among these, DL0609 has the strongest effect, which may be related to its more anti-oxidant groups.Many of the biological actions of flavonoids have been attributed to their antioxidant properties, but the precise mechanisms by which flavonoids exert their beneficial or toxic actions remain unclear. However, their classical hydrogen-donating antioxidant activity is unlikely to be the sole explanation for the cellular effect. We summarized the central effects of related flavonoids and found that dopamine, 5-HT, choline and epinephrine systems were their main targets. In collaboration with our related experimental results, we can draw a conclusion that the prevailing simplistic view of one active compound, one mechanism of action is incorrect. It is more likely that multiple bioactive mechanisms contribute to the anti-Parkinson's activity.